Trypanosoma cruzi antigen, gene encoding therefor and methods of detecting and treating chagas disease

ABSTRACT

The present invention relates to a new Trypanosoma cruzi antigen, called PTc100, and to a code gene  sic! encoding the latter, called Tc100. 
     According to the invention, the nucleotide sequences of Tc100 and the amino acid sequences of PTc100 were determined and described. 
     PTc100 and Tc100, or fragments thereof, modified or otherwise, can be used directly or indirectly for the detection of Trypanosoma cruzi, or for the monitoring of the infection generated by the latter, in man or in animals.

FIELD OF THE INVENTION

The subject of the present invention is a new genetic material encodinga new protein recognized by anti-Trypanosoma cruzi antisera, and itrelates to the use of said gene and protein, especially for diagnostic,pharmaceutical and therapeutic purposes.

BACKGROUND OF THE INVENTION

Trypanosoma cruzi is a flagellate protozoal parasite, a member of theorder Kinetoplastida and of the family Trypanosomatidae, which isresponsible for Chagas disease which affects naturally millions ofpersons, mainly in Latin America.

In vertebrate hosts, Trypanosoma cruzi is present in two forms: onewhich is mobile by means of its flagellum or trypomastigote and whichdoes not divide; the other is aflagellate, or intracellular amastigote,which multiplies by binary division.

Transmission of the protozoan in man occurs through hematophagousinsects of the family Reduviidae, during a blood meal followed bydejections at the site of the bite. The vector insect thus releases theinfectious metacyclic trypomastigote forms which will colonize many celltypes through the blood circulation. Trypanosoma cruzi infects cardiacand skeletal muscular cells, the glial cells and the cells of themononuclear phagocytic system. After passive penetration into the hostcell, the trypomastigote form of the parasite differentiates into theamastigote form, divides actively and then this is followed by a releaseof the trypomastigote forms, thereby causing a new cell invasion.

The insects will complete the parasitic cycle by ingesting, during ablood meal, the trypomastigote forms in the host. The latterdifferentiate into epimastigote forms in the vector's middle intestineand finally into the infectious metacyclic trypomastigote forms in theposterior intestine.

Two phases can be distinguished in the Chagas disease: the acute phaseand the chronic phase. The acute phase occurs after a transfusional,congenital or vectorial type contamination and lasts for a few weeks. Itis characterized by a large number of parasites circulating in the bloodand corresponds to an exponential division of the protozoan. The acutephase is most often asymptomatic. However, in infants contaminated bytheir mother, the acute phase, which is marked by an acute cardiopathy,may be critical. The chronic phase may extend over many years. In someindividuals, this phase is asymptomatic. On the other hand, otherpatients have tissue lesions in the heart or digestive typemanifestations. In any case, clinical diagnosis must always be confirmedby tests for the detection either of antibodies directed against theparasitic antigens, or of the parasite itself.

This disease is becoming a worldwide problem because of thecontamination through blood transfusion. It was therefore becomingessential to have available diagnostic tests which make it possible todetermine the presence of the parasite in individuals. Variousserological tests included direct agglutination, indirectimmunofluorescence (IIF), complement fixation tests (CFR), ELISA tests(Enzyme Linked Immunosorbent Assay). The Trypanosoma cruzi antigens usedfor the serological tests are obtained from a total lysate of thenoninfectious stage of the parasite or from partially purified proteinfractions. However, these fractions do not allow antigens to be obtainedin sufficient quantity and quality for the production of a reliableserological diagnostic test. Furthermore, the complexity of the parasiteand the strain-to-strain antigenic polymorphism introduce an additionaldifficulty in the reproducibility of the different preparations.Finally, there are many risks of cross-reactivity with other protozoa,more particularly with Trypanosoma rangeli, a nonpathogenic parasite,and the family Leishmania. Another disadvantage of these techniques isthe absence of determination of the disease phase which would allow atreatment from the onset of the acute phase.

In order to solve these various problems, it was envisaged to produce aserological diagnostic kit composed of recombinant proteins which wouldbe specific for Trypanosoma cruzi.

Various research groups have screened libraries for expression ofTrypanosoma cruzi genomic DNA or complementary DNA in the vector λgt11,using sera from patients suffering from Chagas disease. The λgt11 phageallows the insertion of foreign DNA of a maximum size of 7 Kb into theEcoR1 site localized in the lacZ gene, under the control of the lacpromoter. The product obtained is a recombinant protein used withbetagalactosidase, which is inducible by IPTG (isopropylbeta-D-thiogalactoside).

Various Trypanosoma cruzi genes, encoding proteins recognized by theChagasic sera were thus characterized. Among the recombinant antigensdescribed, the H49 antigen may be mentioned (Paranhos et al., 1994 (1)).However, this antigen does not allow a serological detection sensitivityof 100% of the patients in the acute or chronic phase. It was thereforeenvisaged to combine the H49 antigen with the CRA antigen (CytoplasmicRepetitive Antigen) (Lafaille et al., (1989) (2)) but still withoutsolving this problem.

SUMMARY OF THE INVENTION

The present inventors have identified and obtained for the first time anew genetic material encoding a new protein, recognized byanti-Trypanosoma cruzi antisera, which makes it possible to overcome theabovementioned disadvantages. The genetic material may be used toproduce proteins or polypeptides for the production of diagnostic tests,or for the preparation of vaccinal or pharmaceutical compositions, ormay itself either be used as a probe, or for the determination ofspecific probes which can be used in nucleic acid hybridization testsfor the detection of Trypanosoma cruzi infections. Likewise, the proteinor any corresponding polypeptide may be used for the production ofantibodies specific for the parasite, for diagnostic or passiveprotection purposes.

This gene was called Tc 100 by the applicant.

DETAILED DESCRIPTION OF THE INVENTION

Consequently, the subject of the present invention is a DNA or RNAmolecule consisting of at least one strand comprising a nucleotidesequence represented in the identifier SEQ ID NO:1, or a sequencecomplementary or antisense or equivalent to said sequence identified inthe identifier SEQ ID NO:1, and especially a sequence having, for anysuccession of 100 contiguous monomers, at least 50%, preferably at least60%, or better still at least 85% homology with said sequence.

Nucleotide sequence is understood to mean either a DNA strand or itscomplementary strand, or an RNA strand or its antisense strand or theircorresponding complementary DNAs. The DNA sequence as represented in theidentifier SEQ ID NO:1 corresponds to the messenger RNA sequence, itbeing understood that the thymine (T) in the DNA is replaced by a uracil(U) in the RNA.

According to the invention, two nucleotide sequences are said to beequivalent in relation to each other, or in relation to a referencesequence if, functionally, the corresponding biopolymers can playessentially the same role, without being identical, with respect to theapplication or use considered, or in the technique in which they areinvolved; two sequences obtained because of the natural variability,especially spontaneous mutation, of the species from which they wereidentified, or because of induced variability, as well as homologoussequences, homology being defined below, are especially equivalent.

Variability is understood to mean any spontaneous or inducedmodification of a sequence, especially by substitution and/or insertionand/or deletion of nucleotides and/or of nucleotide fragments, and/orextension and/or shortening of the sequence at at least one of the ends;a nonnatural variability may result from the genetic engineeringtechniques used; this variability may result in modifications of anystarting sequence, considered as reference, and capable of beingexpressed by a degree of homology relative to the said referencesequence.

Homology characterizes the degree of identity of two nucleotide (orpeptide) fragments compared; it is measured by the percentage identitywhich is especially determined by direct comparison of nucleotide (orpeptide) sequences, relative to reference nucleotide (or peptide)sequences.

Any nucleotide fragment is said to be equivalent to a reference fragmentif it has a nucleotide sequence which is equivalent to the referencesequence; according to the preceding definition, the following areespecially equivalent to a reference nucleotide fragment:

a) any fragment capable of at least partially hybridizing with thecomplementary strand of the reference fragment,

b) any fragment whose alignment with the reference fragment leads to thedetection of identical contiguous bases, in greater number than with anyother fragment obtained from another taxonomic group,

c) any fragment resulting or capable of resulting from the naturalvariability of the species, from which it is obtained,

d) any fragment capable of resulting from the genetic engineeringtechniques applied to the reference fragment,

e) any fragment, containing at least 30 contiguous nucleotides, encodinga peptide homologous or identical to the peptide encoded by thereference fragment,

f) any fragment different from the reference fragment by insertion,deletion, substitution of at least one monomer, extension or shorteningat at least one of its ends; for example any fragment corresponding tothe reference fragment flanked at at least one of its ends by anucleotide sequence not encoding a polypeptide.

The invention moreover relates to DNA or RNA fragments whose nucleotidesequence is identical, complementary, antisense or equivalent to any oneof the following sequences:

that starting at nucleotide 1232 and ending at nucleotide 2207 of SEQ IDNO: 1,

that starting at nucleotide 1232 and ending at nucleotide 1825 of SEQ IDNO:1,

that starting at nucleotide 1266 and ending at nucleotide 2207 of SEQ IDNO; 1,

and especially the DNA or RNA fragments whose sequence has, for anysuccession of 30 contiguous monomers, at least 50%, preferably at least60%, or better still at least 85% homology with any one of saidsequences.

The subject of the invention is also a protein, called PTc100 by theapplicant, having an apparent molecular mass of about 100 kDa, which isrecognized by anti-Trypanosoma cruzi antisera, or an immunologicalequivalent of this protein, and fragments thereof. The amino acidsequence of this protein is represented in the identifier sequence SEQID NO:2.

Immunological equivalent is understood to mean any polypeptide orpeptide capable of being immunologically recognized by the antibodiesdirected against said Ptc100 protein.

The invention also relates to any fragment of the Ptc100 protein. Aparticular protein fragment has a sequence starting at amino acid 323and ending at amino acid 520 of the sequence defined in the identifierSEQ ID NO:2, said fragment being specifically recognized byanti-Trypanosoma cruzi antisera; the invention also relates to anyimmunological equivalent of said fragment.

The Ptc100 protein and said protein fragments may contain modifications,especially chemical modifications, which do not alter theirimmunogenicity.

Moreover, the subject of the present invention is also an expressioncassette which is functional especially in a cell derived from aprokaryotic or eukaryotic organism, and which allows the expression ofDNA encoding the entire Ptc100 protein or a fragment thereof, inparticular of a DNA fragment as defined above, placed under the controlof elements necessary for its expression; said protein and said proteinfragments being recognized by anti-Trypanosoma cruzi antisera.

Generally, any cell derived from a prokaryotic or eukaryotic organismcan be used within the framework of the present invention. Such cellsare known to persons skilled in the art. By way of examples, there maybe mentioned cells derived from a eukaryotic organism, such as the cellsderived from a mammal, especially CHO (Chinese Hamster Ovarian) cells;insect cells; cells derived from a fungus, especially a unicellularfungus or from a yeast, especially of the strain Pichia, Saccharomyces,Schizosaccharomyces and most particularly selected from the groupconsisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe,Schizosaccharomyces malidevorans, Schizosaccharomyces sloofiae,Schizosaccharomyces octosporus. Likewise, among the cells derived from aprokaryotic organism, there may be used, without this constituting alimitation, the cells of a strain of Escherichia coli (E. coli) orenterobacterial cells. A large number of these cells are commerciallyavailable in collections, such as ATCC (Rockville, Ma, USA) and AFRC(Agriculture & Food Research Council, Norfolk, UK). The cell may also beof the wild-type or mutant type. The mutations are described in theliterature accessible to persons skilled in the art.

For the purposes of the present invention, an E. coli DH5a cell(marketed by the company CLONTECH under the reference: C2007-1) is used.

The expression cassette of the invention is intended for the productionof the PTc100 protein or for fragments of said protein which areproduced by the abovementioned E. coli cell, and which are recognized byhuman antisera. Such antisera are obtained from patients who havecontracted a Trypanosoma cruzi infection recently or long ago, andcontain immunoglobulins specifically recognizing PTc100. Of course, thePTc100 protein can also be recognized by other antibodies, such as forexample monoclonal or polyclonal antibodies obtained by immunization ofvarious species with the natural abovementioned protein, the recombinantprotein or fragments or peptides thereof.

PTc100 protein is understood to mean the natural Trypanosoma cruzicytoplasmic antigen, or the antigen produced especially by the geneticrecombination techniques described in the present application, or anyfragment or mutant of this antigen, provided that it is immunologicallyreactive with antibodies directed against the PTc100 protein of thisparasite.

Advantageously, such a protein has an amino acid sequence having adegree of homology of at least 70%, preferably of at least 85%, and mostpreferably of at least 95% relative to the sequence identified in theidentifier SEQ ID NO:2. In practice, such an equivalent can be obtainedby deletion, substitution and/or addition of one or more amino acids ofthe native or recombinant protein. It is within the capability ofpersons skilled in the art to perform, using known techniques, thesemodifications without affecting immunological recognition.

Within the framework of the present invention, the PTc100 protein can bemodified in vitro, especially by deletion or addition of chemicalgroups, such as phosphates, sugars or myristic acids, so as to enhanceits stability or the presentation of one or several epitopes.

The expression cassette according to the invention allows the productionof a PTc100 protein (having an amino acid sequence as specified above)and fragments of said protein, fused with an exogenous element which canhelp its stability, its purification, its production or its recognition.The choice of such an exogenous element is within the capability ofpersons skilled in the art. It may be especially a hapten, an exogenouspeptide or a protein.

The expression cassette according to the invention comprises theelements necessary for the expression of said DNA fragment in the cellconsidered. "Elements necessary for the expression" is understood tomean the elements as a whole which allow the transcription of the DNAfragment into messenger RNA (mRNA) and the translation of the latterinto protein.

The present invention also extends to a vector comprising an expressioncassette according to the invention. This may be a viral vector andespecially a vector derived from a baculovirus, more particularlyintended for expression in insect cells, or an adeno-virus-derivedvector for expression in mammalian cells.

It may also be an autonomously replicating plasmid vector and inparticular a multiplicative vector.

The present invention also relates to a cell derived from a prokaryoticor eukaryotic organism, comprising an expression cassette, either in aform integrated in the cellular genome, or inserted in a vector. Such acell was previously defined.

The subject of the present invention is also a process for preparing aPTc100 protein, or fragments of said protein, according to which:

(i) a cell derived from a prokaryotic or eukaryotic organism, comprisingthe expression cassette according to the invention, is cultured underappropriate conditions; and

(ii) the expressed protein derived from the abovementioned organism isrecovered.

The present invention also relates to one or more peptides, whose aminoacid sequence corresponds to a portion of the sequence of the PTc100protein and exhibiting, alone or as a mixture, a reactivity with theentire sera from individuals or animals infected with Trypanosoma cruzi.

The peptides can be obtained by chemical synthesis, lysies of the PTc100protein or by genetic recombination techniques.

The invention also relates to monoclonal or polyclonal antibodiesobtained by immunological reaction of a human or animal organism to animmunogenic agent consisting of the natural or recombinant PTc100protein and fragments thereof, or of a peptide, as defined above.

The present invention also relates to a reagent for the detection and/ormonitoring of a Trypanosoma cruzi infection, which comprises, asreactive substance, a PTc100 protein as defined above, or fragmentsthereof, a peptide or a mixture of peptides as defined above, or atleast one monoclonal or polyclonal antibody as described above.

The above reagent may be attached directly or indirectly to anappropriate solid support. The solid support may be especially in theform of a cone, a tube, a well, a bead and the like.

The term "solid support" as used here includes all materials on which areagent can be immobilized for use in diagnostic tests. Natural orsynthetic materials, chemically modified or otherwise, can be used assolid supports, especially polysaccharides such as cellulose-basedmaterials, for example paper, cellulose derivatives such as celluloseacetate and nitrocellulose; polymers such as vinyl chloride,polyethylene, polystyrenes, polyacrylate or copolymers such as polymersof vinyl chloride and propylene, polymers of vinyl chloride and vinylacetate; styrene-based copolymers, natural fibers such as cotton andsynthetic fibers such as nylon.

Preferably, the solid support is a polystyrene polymer or abutadiene/styrene copolymer. Advantageously, the support is apolystyrene or a styrene-based copolymer comprising between about 10 and90% by weight of styrene units.

The binding of the reagent onto the solid support may be performed in adirect or indirect manner.

Using the direct manner, two approaches are possible: either byadsorption of the reagent onto the solid support, that is to say bynoncovalent bonds (principally of the hydrogen, Van der Walls or ionictype), or by formation of covalent bonds between the reagent and thesupport. Using the indirect manner, an "anti-reagent" compound capableof interacting with the reagent so as to immobilize the whole onto thesolid support can be attached beforehand (by adsorption or covalentbonding) onto the solid support. By way of example, there may bementioned an anti-PTc100 antibody, on the condition that it isimmunologically reactive with a portion of the protein different fromthat involved in the reaction for recognizing the antibodies in thesera; a ligand-receptor system, for example by grafting onto the PTc100protein a molecule such as a vitamin, and by immobilizing onto the solidphase the corresponding receptor (for example the biotin-streptavidinsystem). Indirect manner is also understood to mean the preliminarygrafting or fusion by genetic recombination of a protein, or a fragmentof this protein, or of a polypeptide, to one end of the PTc100 protein,and the immobilization of the latter onto the solid support by passiveadsorption or covalent bonding of the protein or of the polypeptidegrafted or fused.

The invention also relates to a process for the detection and/ormonitoring of a Trypanosoma cruzi infection in a biological sample, suchas a blood sample from an individual or an animal likely to have beeninfected with Trypanosoma cruzi, characterized in that said sample and areagent as defined above are placed in contact, under conditionsallowing a possible immunological reaction, and the presence of animmune complex with said reagent is then detected.

By way of non-limiting example, there may be mentioned the sandwich-typedetection process in one or more stages, as especially described inpatents FR 2,481,318 and FR 2,487,983, which consists in reacting afirst monoclonal or polyclonal antibody specific for a desired antigen,attached onto a solid support, with the sample, and in revealing thepossible presence of an immune complex thus formed using a secondantibody labelled by any appropriate marker known to persons skilled inthe art, especially a radioactive isotope, an enzyme, for exampleperoxidase or alkaline phosphatase and the like, using so-calledcompetition techniques well known to persons skilled in the art.

The subject of the invention is also an active immunotherapeuticcomposition, especially a vaccinal preparation, which comprises asactive ingredient, a natural or recombinant PTc100 protein or fragmentsthereof, or the peptides identified above, the active ingredient beingoptionally conjugated with a pharmaceutically acceptable carrier, andoptionally an excipient and/or an appropriate adjuvant.

The present invention also covers a pharmaceutical composition intendedfor the treatment or for the prevention of a Trypanosoma cruzi infectionin man or in an animal, comprising a therapeutically effective quantityof an expression cassette, a vector, a cell derived from a prokaryoticor eukaryotic organism as defined above, a PTc100 protein according tothe invention, or fragments thereof, or an antibody of the invention.

The subject of the present invention is also probes and primers specificfor T. cruzi, and their uses in diagnostic tests.

The term probe as used in the present invention refers to a DNA or RNAcontaining at least one strand having a nucleotide sequence which allowshybridization to nucleic acids having a nucleotide sequence asrepresented in the identifier SEQ ID NO:1, or a complementary orantisense sequence, or a sequence equivalent to said sequence, andespecially a sequence having, for any succession of 5 to 100 contiguousmonomers, at least 50%, preferably at least 60%, or even better at least85% homology with SEQ ID NO:1, with fragments thereof, or with asynthetic oligonucleotide allowing such a hybridization, nonmodified orcomprising one or more modified bases such as inosine,5-methyldeoxycytidine, deoxyuridine, 5-dimethylaminodeoxyuridine,2,6-diaminopurine, 5-bromodeoxyuridine or any other modified base.Likewise, these probes may be modified at the level of the sugar, namelythe replacement of at least one deoxyribose with a polyamide (P. E.NIELSEN et al. (1991) (13)), or at the level of the phosphate group, forexample its replacement with esters, especially chosen from esters ofdiphosphate, of alkyl and arylphosphonate and of phosphorothioate.

The probes may be much shorter than the sequence identified in theidentifier SEQ ID NO:1. In practice, such probes comprise at least 5monomers, advantageously from 8 to 50 monomers, having a hybridizationspecificity, under defined conditions, to form a hybridization complexwith DNA or RNA having a nucleotide sequence as defined above.

A probe according to the invention can be used for diagnostic purposesas capture and/or detection probe, or for therapeutic purposes.

The capture probe can be immobilized on a solid support by anyappropriate means, that is to say directly or indirectly, for example bycovalent bonding or passive adsorption.

The detection probe is labelled by means of a marker chosen fromradioactive isotopes, enzymes especially chosen from peroxidase andalkaline phosphatase, and those capable of hydrolyzing a chromogenic,fluorigenic or luminescent substrate, chromophoric chemical compounds,chromogenic, fluorigenic or luminescent compounds, nucleotide baseanalogs, and biotin.

The probes of the present invention which are used for diagnosticpurposes can be used in any known hybridization technique, andespecially the so-called "Dot-Blot" technique (Maniatis et al. (1982)(14)), the Southern Blotting technique (Southern E. M. (1975) (15)), theNorthern Blotting technique, which is a technique identical to theSouthern Blotting technique but which uses RNA as target, and thesandwich technique (Dunn A. R. et al. (1977) (16)). Advantageously, thesandwich technique is used which comprises a specific capture probeand/or a specific detection probe, it being understood that the captureprobe and the detection probe must have a nucleotide sequence which isat least partially different.

Another application of the invention is a therapeutic probe for treatinginfections due to Trypanosoma cruzi, said probe being capable ofhybridizing in vivo with the DNA or RNA of the parasite to block thetranslation and/or transcription and/or replication phenomena.

A primer is a probe comprising 5 to 30 monomers, having a hybridizationspecificity, under predefined conditions, for the initiation of anenzymatic polymerization, for example in an amplification technique suchas PCR (Polymerase Chain Reaction), in an elongation process such assequencing, in a reverse transcription method and the like.

A preferred probe or primer will contain a nucleotide sequence chosenfrom the sequences SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:12.

The invention also relates to a reagent for detecting and/or identifyingTrypanosoma cruzi in a biological sample, comprising at least one probeas defined above, and in particular a capture probe and a detectionprobe, either or both corresponding to the above definition.

The invention therefore provides a process for selectively detectingand/or for identifying Trypanosoma cruzi in a biological sample,according to which the RNA, extracted from the parasite and optionallydenatured, or the DNA, denatured extract, or the DNA obtained fromreverse transcription of the RNA, is exposed to at least one probe asdefined above and the hybridization of said probe is detected.

The invention will be understood more clearly upon reading the detaileddescription below which is made with reference to the accompanyingfigures in which:

DESCRIPTION OF THE FIGURES

FIG. 1 represents the restriction map of the Tc100 gene, which map isdeduced by Southern blotting of different fragments obtained afterdigestion of Trypanosoma cruzi DNA with restriction endonucleases.

FIG. 2 is a schematic representation of the three overlapping regions ofthe Tc100 cDNA. The numbered arrows represent the oligonucleotides usedas primers for the PCR amplification.

EXAMPLE 1

Isolation of the Tc50 clone

An expression library was constructed from Trypanosoma cruzi genomic DNAfragments. The T. cruzi, strain G (YOSHIDA. N, (1983) (17)), DNAisolated from the metacyclic trypomastigote stage was digested with theenzyme DNase I. After selection of the fragments according to theirsize, they were ligated to synthetic EcoRI adaptors and cloned into theEcoRI site of lambda gt11 vector DNA (Young and Davis, 1983 (3); Cotrimet al., 1990) (4).

The clone, called Tc50 by the applicant, was isolated from the libraryby immunological screening with the aid of a mixture of sera frompatients suffering from the chronic phase of the Chagas disease.

The Tc50 phage clone was purified, amplified and the insert was detectedby the PCR ("Polymerase Chain Reaction") technique with the aid of theprimers: ##STR1## corresponding respectively to the nucleotide sequenceof the left and right arms of the lambda gt11 phage DNA.

The 594 base pairs (bp) Tc50 DNA fragment, after EcoRI digestion, wassubcloned into the expression vector pGEX (Pharmacia) linearized withEcoRI. The sequencing of the Tc50 clone DNA was carried out in this samevector with the aid of specific primers situated in 3' and 5' of thecloning site of pGEX, according to the chain termination technique(Sanger et al., 1977 (5)) and according to the manufacturer's procedure(USB-Amersham).

The nucleotide sequence of the 594 bp Tc50 fragment as well as itsdeduced amino acid sequence (198 aa) are represented in the identifiersSEQ ID NO:1 and SEQ ID NO:2, respectively. The nucleotide sequence ofthe 594 bp Tc50 fragment starts at nucleotide (nt) 1232 and ends atnucleotide 1825 of SEQ ID NO:1. The corresponding amino acid sequencestarts at amino acid 323 and ends at amino acid 520 of SEQ ID NO:2.

EXAMPLE 2

Expression of the Tc50 clone in Escherichia coli

The construct pGEX-Tc50 (198 aa) synthesizes, in the bacterium DH5alpha,a protein fused with GST ("Glutathione S Transferase"), with an apparentmolecular mass of 50 kDa, which is detected by SDS-PAGE polyacrylamidegel electrophoresis (SDS: sodium dodecyl sulfate) (Laemmli, 1970 (6)).The reactivity of the protein towards chagasic human sera was confirmedby the Western blotting technique (Towbin et al., 1979 (7)) with the aidof the same mixture of chronic phase chagasic sera which is used forscreening the lambda gt11 library.

The soluble fraction of the recombinant GST-Tc50 protein obtained afterlysis of the bacterial extracts by ultrasound was purified by affinitychromatography on a glutathione agarose column (Sigma), according to themethod of Smith and Johnson, (1988) (8).

The antigenic properties of the recombinant GST-Tc50 antigen were testedby ELISA (Voller et al., 1975 (9)). Microtiter plates (Maxisorp (tradename) were sensitized with 100 ng/ml of GST-Tc50 antigen in 100 mMNaHCO3 (pH 9.6). After incubation with the patients' sera, the immunecomplexes were detected with the aid of a peroxidase-coupled antihumanIgG goat serum.

The results are presented in the accompanying table and show that thechagasic human sera tested reacts specifically with the recombinantprotein. No cross-reactivity was observed on 7 sera from patientssuffering from cutaneous or visceral leishmaniosis.

EXAMPLE 3

Identification of the native T. cruzi protein having the antigenicdeterminants of the Tc50 clone

The detection of the native T. cruzi protein was performed afterimmunopurification of a mixture of chagasic human sera on thecorresponding recombinant protein called PTc50 by the applicant.

The eluate of monospecific polyclonal antibodies which is obtained wasused as probe, in Western blotting, on total protein extracts ofdifferent stages of the parasite. The selected antibodies specificallyreacted with a protein of apparent molecular mass 100 kDa, called PTc100by the applicant, which is expressed in all the tested strains of theparasite.

EXAMPLE 4

Molecular analysis of the Tc100 gene-Southern blots

In order to establish the restriction map of the Tc100 gene (FIG. 1),the T. cruzi, strain G, nuclear DNA was digested with differentrestriction endonucleases (BamHI, EcoRI, HindIII, PstI, PvuII, SacI,BamHI/EcoRI, BamHI/PvuII, EcoRI/HindIII, EcoRI/PstI, EcoRI/PvuII,EcoRI/SacI, PstI/SacI, PstI/PvuII, PvuII/SacI, PvuII/HindIII), separatedon agarose gel and then transferred onto a nylon filter according to theSouthern technique. The Southern blot hybridization was performed withthe 594 bp Tc50 DNA, which is a fragment of the Tc100 DNA describedabove, radiolabelled with ³² P by random incorporation (Amersham).

Cloning of a 3500 bp Tc100 genomic fragment

According to the results obtained by Southern blotting, the T. cruzi,strain G, genomic DNA was digested with the enzyme EcoRI and thenseparated on agarose gel. The EcoRI restriction fragments of about 3500bp (FIG. 1) were cloned into the vector lambda gt10 (Huynh et al., 1984(10)) linearized by EcoRI. The phage clone containing the 3500 bp Tc100genomic insert was isolated with the aid of the 594 bp radiolabelledprobe described above. A 1041 bp fragment situated in the 3' region ofthe 3500 bp Tc100 genomic insert was sequenced. This sequencing wascarried out gradually with the aid of the following primers: ##STR2##

The primer SEQ ID NO:5 is situated in the previously sequenced portionof the 594 bp Tc50 fragment. The primer SEQ ID NO:6 corresponds to thelambda gt10 phage primer.

This 1041 bp fragment, which starts at nucleotide 1403 and ends atnucleotide 2443 of SEQ ID NO:1, has an open reading frame in phase withthe sequence of the 594 bp Tc50 fragment.

EXAMPLE 5

Cloning of the Tc100 cDNA

The cDNA was synthesized from total RNA from T. cruzi, strain G,epimastigots. The Tc100 cDNA was amplified by the PCR technique in threedifferent fragments: a fragment A corresponding to the 5' region of 1459bp, a fragment B corresponding to the central region of 942 bp, afragment C corresponding to the 3' region of 1406 bp of the Tc100 cDNA,as schematically represented in FIG. 2.

Cloning of fragment A of the Tc100 cDNA

The total cDNA synthesized by AMV ("avian myeloblastosis virus") reversetranscriptase, with the aid of random hexanucleotides (BoehringerMannheim), was amplified, by PCR, using the following pair of primers:##STR3##

SEQ ID NO:7 corresponds to a portion of the consensus sequence of 35nucleotides present in 5' of the mRNAs in trypanosomatides and called"spliced leader" (Parsons et al. 1984 (11)).

SEQ ID NO:8 corresponds to the sequence complementary to a portion ofthe predetermined sequence of the 594 bp fragment, and starts atnucleotide 1442 and ends at nucleotide 1459 of SEQ ID NO:1, according tothe coding strand numbering.

After verification by Southern blotting with the aid of theradiolabelled 594 bp probe previously described, the 1459 bp cDNAfragment corresponding to the 5' region of Tc100 was cloned into theplasmid called pCRII (trade name) (Invitrogen), and sequenced. Thesequence represented in the identifier SEQ ID NO:7 starts at nucleotide1 and ends at nucleotide 1459.

Cloning of fragment B of the Tc100 cDNA

The T. cruzi total cDNA was amplified by PCR with the aid of theprimers: ##STR4##

The sequence ID NO:9 which corresponds to a portion of the 594 bppredetermined sequence of the Tc100 gene starts at nucleotide 1266 andends at nucleotide 1287 of SEQ ID NO:7.

The sequence SEQ ID NO:10 corresponds to the sequence complementary to aportion of the 1041 bp previously described sequence of the Tc100 gene.This sequence SEQ ID NO:10 starts at nucleotide 2187 and ends atnucleotide 2207 of SEQ ID NO:1, according to the coding strandnumbering.

The fragment obtained, 942 bp in length, was cloned into the plasmidpCRII and sequenced. The sequence represented in the identifier SEQ IDNO:7 starts at nucleotide 1266 and ends at nucleotide 2207.

Cloning of fragment C of the Tc100 cDNA

In order to isolate the 3' portion of the Tc100 cDNA, the T. cruzi totalcDNA was synthesized with the aid of the adaptor oligo(dT)₁₆ hybridprimer. ##STR5## according to the RACE ("Rapid Amplification of cDNAEnds") procedure (Frohman et., 1988 (12)).

The 3' region of the Tc100 cDNA was amplified using the adaptor primerand the following pair of primers: ##STR6##

The sequence SEQ ID NO:12 corresponds to a portion of the previouslydescribed 1041 bp sequence of the Tc100 gene, starting at nucleotide1997 and ending at nucleotide 2017.

The sequence SEQ ID NO:11 corresponds to the arbitrary sequence of theadaptor represented in SEQ ID NO:11.

After checking by Southern blotting using the 1041 bp radiolabelledfragment previously described, the 3' fragment of the Tc100 cDNA, 1423bp long, was cloned into pCRII and sequenced. The sequence representedin the identifier SEQ ID NO:7 starts at nucleotide 1997 and ends atnucleotide 3402.

The Tc100 complete cDNA, 3402 bp in size, was completely sequenced. Ithas a 2745 bp open reading frame and the deduced amino acid sequence is915. The methionine codon is in position 266 and the stop codon inposition 3011.

The Trypanosoma cruzi Tc100 gene encodes the new PTc100 protein oftheoretical molecular mass 100 kDa.

Of course, since the DNA sequence of the gene has been fully identified,it is possible to produce the corresponding DNA solely by chemicalsynthesis, and then to insert the DNA into commercially available DNAvectors, using known techniques from the technology relating to geneticrecombination.

                  TABLE                                                           ______________________________________                                                                    OD (492 nm)                                                                   detection                                         Disease             Sera    threshold = 0.320                                 ______________________________________                                        CHAGAS DISEASE       1      1.358 (+)                                                              2      1.278 (+)                                                              3      0.328 (+)                                                              4      0.404 (+)                                                              5      1.378 (+)                                                              6      1.059 (+)                                                              7      0.895 (+)                                                              8      1.791 (+)                                                              9      1.635 (+)                                                             10      1.427 (+)                                                             11      1.009 (+)                                                             12      1.743 (+)                                                             13      0.530 (+)                                                             14      1.035 (+)                                                             15      0.461 (+)                                         CUTANEOUS LEISHMANIOSIS                                                                           16      0.291 (-)                                         VISCERAL LEISHMANIOSIS                                                                            17      0.071 (-)                                         (Kala azar)         18      0.081 (-)                                                             19      0.279 (-)                                                             20      0.098 (-)                                                             21      0.067 (-)                                                             22      0.125 (-)                                         ______________________________________                                    

BIBLIOGRAPHIC REFERENCES

Conventional molecular biology techniques were performed according tothe procedures cited in: "Molecular cloning, a laboratory manual".Maniatis T., Fritsch E. F. & Sambrook J. Second edition. Cold SpringHarbor Laboratory Press (New York) (1989).

1. Paranhos-Baccala G., Santos M., Cotrim P., Rassi A., Jolivet M.,Camargo M. E. & Da Silveira J. F. Detection of antibodies in sera fromChagas disease patients using a Trypanosoma cruzi immunodominantrecombinant antigen. Parasite Immunology (1994): 16: 165-169.

2. Lafaille J. J., Linss J., Krieger M. A., Padron T. S., De Souza W &Goldenberg S. Structure and expression of two Trypanosoma cruzi genesencoding antigenic proteins bearing repetitive epitopes. Molecular andBiochemical Parasitology. (1989). 35 : 127-136.

3. Young R. A. & Davis R. W. Efficient isolation of genes by usingantibody probes. Proc. Natl. Acad. Sci. USA. (1983). 80 : 1194-1198.

4. Cotrim P. C., Paranhos G., Mortara R. A., Wanderley J., Rassi.,Camargo ME. & Da Silveira J. F. Expression in Escherichia coli of adominant immunogen of Trypanosoma cruzi recognized by human chagasicsera. Journal of Clinical Microbiology. (1990). 28(3): 519-524.

5. Sanger F., Nicklen S. & Coulson A. R. DNA sequencing withchain-terminating inhibitors. Proc. Natl. Acad. Sci. USA. (1977). 74 :5463-5467.

6. Laemmli U. K. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature. (1970). 227 : 680-685.

7. Towbin H., Staehelin T. & Gordon J. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedureand some applications. Proc. Natl. Acad. Sci. USA. (1979). 76 :4350-4354.

8. Smith D. B. & Johnson K. S. Single step purification of polypeptidesexpressed in Escherichia coli as fusions with glutathione S transferase.Gene. (1988). 67 : 31-40.

9. Voller A., Draper C., Bidwell D. E. & Bartlett A. Microplateenzyme-linked immunosorbent assay for Chagas disease. Lancet (1975). 1.:1426.

10. Huynh T. V., Young R. A. & Davis R. W. DNA Cloning: A practicalapproach. (1984) (Ed. D. Glover) 49-78. IRL. Oxford.

11. Parsons M., Nelson R. G., Watkins K. P. & Agabian N. TrypanosomemRNAs share a common 5' Spliced Leader sequence. Cell. (1984). 38 :309-316.

12. Frohman M. A., Dush M. K. & Martin G. R. Rapid production of fulllengh sic! cDNA from rare trans-cripts: amplification using a singlegene specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA. (1984)85 : 8998-9002.

13. Nielsen P. E. et al., Science, 254, 1497-1500 (1991).

14. Maniatis et al., Molecular Cloning, Cold Spring Harbor (1982).

15. Southern E. M., J. Mol. Biol., 98, 503 (1975).

16. Dunn A. R., Hassel J. A., Cell, 12, 23 (1977).

17. Yoshida N, Surface antigens of metacyclic trypomastigotes ofTrypanosoma cruzi, Infection and Immunity, 40, 386-389, (1983).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 13                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3402 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AACGCTATTATTAGAACAGTTTCTGTACTATATTGTCATTTGGGGAGGGGGGAAAGGGGG60                GAAGTACTTGCCGTTTTGTGTGGGTGACGAGACAACACACATCGAGCGGGAAGAAAAAAA120               AAAAGGAAATAAATTAAATTAAATTATTTGTTCTTTGAATAGGCAAAGAAGAAGAAGAAG180               AAAAGGTGCGGGGGAGGGAGGAGAAAGCGACACACACACAAAAAAAAAAAAAGGAATTGC240               GGAAATAACAACGCAAGGCGCGGACATGACCGTGACGGTGGATTTGTTCAATCATGCGAA300               GCCGAGCAACAATGAGGGCCGCGTGTGGTCTGTGGACGCCGCGACATTTAACGAGGTGCC360               TGAGGCGCAGCGTGTGCTGGCGGATTCGCAGTTTTATCTTGCCTACACCATGAAGCGGCG420               TCACGTGCTGCGTGTGGTGAAGCGCTCGAACCTTTTGAAGGGCACCGTGCGGGCACACTC480               AAAGCCCATTCATGCGGTGAAGTTTGTGAATTACCGCAGTAACGTCGCAGCATCGGCTGG540               GAAGGGGGAGTTCTTCGTGTGGGTTGTGACGGATGAAACGGAGGCGAGCAACGGCAAGCC600               GGATCTCGCAGCCCGCCTCACAGTGAAGGTGTACTTTAAGCTTCAGGATCCTGTCACAAT660               TCCATGCTTTTCTTTCTTTATCAACGCCGAGAGTCAGCGGCCTGATCTGCTTGTCCTTTA720               CGAAACGCAGGCGGCAATTCTTGACAGCTCCTCCCTCATTGAGCGCTTTGACGTGGAATC780               ACTGGAGGCAACACTACAGCGGAATTGCACAACCCTGCGAACCCTGACTCAACCGGTTAG840               TGAGAACAGTTTATGCTCCGTTGGCTCTGGCGGATGGTTCACCTTTACCACGGAACCAAC900               AATGGTAGCGGCATGCACATTACGAAACCGCAGCACTCCATCATGGGCGTGTTGCGAGGG960               TGAGCCAGTGAAGGCATTGCATCTCCTTGACGCAACCGTTGAGGAAAATGTCAGTGTTCT1020              CGTGGCCGCATCTACAAAAGGGGTGTACCAATGGCTCCTTACGGGTGTAGCAGAACCAAA1080              CTTGTTGCGCAAGTTTGTCATTGATGGATCTATTGTCGCGATGGAAAGCTCACGAGAAAC1140              GTTTGCCGTGTTTGACGACAGGAAGCAGCTGGCGCTGGTCAACATGCATTCCCCTCATAA1200              CTTTACCTGCACACACTACATGATGCCTTGTCAGGTACAGCGTAACGGCTTTTGCTTCAA1260              TCGTACAGCCGACGGTAGCTGCGTCCTGGCTGACATGTCGATTCGATTGACGATCTTCCA1320              TCTCCGGTCCTCCCGCAGGGAAGAACAGCAGCCAGGCCAAAAAACATCGGTAGTGGCGAC1380              GGCGAAACCGGGGTGTGTGTCCTCGGGCACTGACGCGGCGAGTAGCAGTCATACCAATAC1440              GACTTCTGCCGCTGCTGCATCCCCTGCATCACCCCCTGTTTCAGCGCCAGCCAAGGCAGC1500              CGCGCCTCCTGCCGCGGCGCGATCGGCTGAGCCGCACGTGGGGAGCAAGATCATTGCTAA1560              TCTAGTGAATCAGCTGGGGATTAATGTCACCCAAAGGAGCGTCGTCAGCACTGGAGCGCC1620              GGCCACGACGAGGTCTACGGCGGTGACGTCCACGACTACCGCCCCGCAGCGAACAAGTCC1680              ATACGGGCACAATGGCCGACCTGTGACGGCTGGATTGGTGGCAGCTAATAGTGGTGCCAG1740              CGCGGCCTCGTCTCCCACAGCCGCGGCGAAACCAACAGGAGAAGAAAAGGCCTCCGCGGC1800              ATGTGAAACGAGCTCCGTGGCGATAAATGCGACACGCCCGGCGCTTCACAACGCCTCTCT1860              CCCGCAGGCGCCAACGGATGGCGTTTTGGCGGCAGCAGTATACCAGTCGGAGGGCGAGGT1920              TCATCAGTCGCTGGAGCGGCTGGAGTCCGTCATAACCAACACGTCTCGGGTTCTGAAGTT1980              GCTCCCTGACACCATTCGAAGAGACCATGAACAACTTCTGAATCTGGGTTTAGAGGCACA2040              GATGACAGAGCTGCAGCAGAGCCGTCCAACACCGCAAACACAGCCGAGAGACACAAGCTC2100              CGCGAAATCATCCGTGTTTGAGACGTACACCCTTGTTCTCATTGCGGATTCCCTCTCTCG2160              CAACATCACGAAGGGGGTGAAGCGTGGTGTGAACGAGGCCATTATGTTGCATCTCGACCA2220              TGAGGTGCGGCACGCCATAGGGAACCGGCTTCGGCAAACACAAAAGAACATCATCAAGAG2280              CCGCCTCGATGAAGCGTTGAAGGAAAGCACTACACAGTTTACGGCTCAATTGACGCAAAC2340              GGTGGAGAATCTGGTGAAGCGCGAGCTTGCCGAGGTGCTTGGTAGCATCAACGGCTCCCT2400              CACTTCTCTCGTGAAGGAAAATGCCTCATTACAGAAAGAGTTGAATTCCATAATGTCTAG2460              TGGGGTGTTGGATGAAATGCGTCGTATGCGGGAAGAGCTGTGCACATTGCGAGAGTCCGT2520              TGCGAAGCGGAAGGCAACAATGCCAGATTCTTCTCTTCACGCCACGAGCTCCTTTCAAGG2580              AAGAAGGTCTGCGCCCGAGACAATTCTTGCAACCGCGTTATCGATGGTGCGAGAGCAGCA2640              ATACCGTCAGGGACTGGAATACATGTTGATGGCTCAGCAGCCCTCTCTCCTCCTGCGGTT2700              CCTCAGCATACTTACAAGGGAAAACGAAAACGCCTACTCGGAACTTATTGAAAATGTAGA2760              GACGCCGAATGACGTGTGGTGTTCGGTTCTGTTGCAACTCATAGAGGCCGCGGCGACCGA2820              GGCTGAGAAGGAGGTGGTTGTTGGCGTCGCCATTGATATTCTCTCCGAGCGCGATCAAAT2880              TGCTCAGAACGGCGCACTCGGCTCGAAACTCACCACCGCCATGCGAGCCTTTGAGCGACA2940              GGCAAGGTCGGAGACAACGAGCAGGTCATTCTTGCAATGCCTGAAGAACCTGGAAAAGCT3000              TCTGCAATCATGATAATAAAAAGAACTCAACGAATACAGTTGTTGATTATTAAGGAAGGG3060              AAAAGAGAGAAAGAGAGAGAGAGAGAGAGAAATGTAATGGGCGTTTAGTTACGGTAGAAA3120              GAAAACGTGTGGATAAGAAGGAGGGGTTTTGTGTGCGACCAGGAATTACTGGGGAACGCT3180              GCTACACGGCGGAATCGACCATTTTATTATTATTATTATTGTCTTTAGTATTATGTTTTT3240              TCTTGTGTGTGTGTGTGTGTGTTTGTGTGTGTGCGGTTATTTTGTATCCGTTTGCTCCCG3300              CCCCTGCCCCCCATCACCCGAGGAGAAAGTAGAATAAGACACATACGATTGTTGTTTTTG3360              TTATCCTTAAAAGGAAGAGAGACCAAAAAAAAAAAAAAAAAA3402                                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 915 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "protein"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetThrValThrValAspLeuPheAsnHisAlaLysProSerAsnAsn                              151015                                                                        GluGlyArgValTrpSerValAspAlaAlaThrPheAsnGluValPro                              202530                                                                        GluAlaGlnArgValLeuAlaAspSerGlnPheTyrLeuAlaTyrThr                              354045                                                                        MetLysArgArgHisValLeuArgValValLysArgSerAsnLeuLeu                              505560                                                                        LysGlyThrValArgAlaHisSerLysProIleHisAlaValLysPhe                              65707580                                                                      ValAsnTyrArgSerAsnValAlaAlaSerAlaGlyLysGlyGluPhe                              859095                                                                        PheValTrpValValThrAspGluThrAspAlaSerAsnGlyLysPro                              100105110                                                                     AspLeuAlaAlaArgLeuThrValLysValTyrPheLysLeuGlnAsp                              115120125                                                                     ProValThrIleProCysPheSerPhePheIleAsnAlaGluSerGln                              130135140                                                                     ArgProAspLeuLeuValLeuTyrGluThrGlnAlaAlaIleLeuAsp                              145150155160                                                                  SerSerSerLeuIleGluArgPheAspValGluSerLeuGluAlaThr                              165170175                                                                     LeuGlnArgAsnCysThrThrLeuArgThrLeuThrGlnProValSer                              180185190                                                                     GluAsnSerLeuCysSerValGlySerGlyGlyTrpPheThrPheThr                              195200205                                                                     ThrGluProThrMetValAlaAlaCysThrLeuArgAsnArgSerThr                              210215220                                                                     ProSerTrpAlaCysCysGluGlyGluProValLysAlaLeuHisLeu                              225230235240                                                                  LeuAspAlaThrValGluGluAsnValSerValLeuValAlaAlaSer                              245250255                                                                     ThrLysGlyValTyrGlnTrpLeuLeuThrGlyValAlaGluProAsn                              260265270                                                                     LeuLeuArgLysPheValIleAspGlySerIleValAlaMetGluSer                              275280285                                                                     SerArgGluThrPheAlaValPheAspAspArgLysGlnLeuAlaLeu                              290295300                                                                     ValAsnMetHisSerProHisAsnPheThrCysThrHisTyrMetMet                              305310315320                                                                  ProCysGlnValGlnArgAsnGlyPheCysPheAsnArgThrAlaAsp                              325330335                                                                     GlySerCysValLeuAlaAspMetSerAsnArgLeuThrIlePheHis                              340345350                                                                     LeuArgCysSerArgArgGluGluGlnGlnProGlyGlnLysThrSer                              355360365                                                                     ValValAlaThrAlaLysProGlyCysValSerSerGlyThrAspAla                              370375380                                                                     AlaSerSerSerHisThrAsnThrThrSerAlaAlaAlaAlaSerPro                              385390395400                                                                  AlaSerProProValSerAlaProAlaLysAlaAlaAlaProProAla                              405410415                                                                     AlaAlaArgSerAlaGluProHisValGlySerLysIleIleAlaAsn                              420425430                                                                     LeuValAsnGlnLeuGlyIleAsnValThrGlnArgSerValValSer                              435440445                                                                     ThrGlyAlaProAlaThrThrArgSerThrAlaValThrSerThrThr                              450455460                                                                     ThrAlaProGlnArgThrSerProTyrGlyHisAsnGlyArgProVal                              465470475480                                                                  ThrAlaGlyLeuValAlaAlaAsnSerGlyAlaSerAlaAlaSerSer                              485490495                                                                     ProThrAlaAlaAlaLysProThrGlyGluGluLysAlaSerAlaAla                              500505510                                                                     CysGluThrSerSerValAlaIleAsnAlaThrArgProAlaLeuHis                              515520525                                                                     AsnAlaSerLeuProGlnAlaProThrAspGlyValLeuAlaAlaAla                              530535540                                                                     ValTyrGlnSerGluGlyGluValHisGlnSerLeuGluArgLeuGlu                              545550555560                                                                  SerValIleThrAsnThrSerArgValLeuLysLeuLeuProAspThr                              565570575                                                                     IleArgArgAspHisGluGlnLeuLeuAsnLeuGlyLeuGluAlaGln                              580585590                                                                     MetThrGluLeuGlnGlnSerArgProThrProGlnThrGlnProArg                              595600605                                                                     AspThrSerSerAlaLysSerSerValPheGluThrTyrThrLeuVal                              610615620                                                                     LeuIleAlaAspSerLeuSerArgAsnIleThrLysGlyValLysArg                              625630635640                                                                  GlyValAsnGluAlaIleMetLeuHisLeuAspHisGluValArgHis                              645650655                                                                     AlaIleGlyAsnArgLeuArgGlnThrGlnLysAsnIleIleLysSer                              660665670                                                                     ArgLeuAspGluAlaLeuLysGluSerThrThrGlnPheThrAlaGln                              675680685                                                                     LeuThrGlnThrValGluAsnLeuValLysArgGluLeuAlaGluVal                              690695700                                                                     LeuGlySerIleAsnGlySerLeuThrSerLeuValLysGluAsnAla                              705710715720                                                                  SerLeuLysLysGluLeuAsnSerIleMetSerSerGlyValLeuAsp                              725730735                                                                     GluMetArgArgMetArgGluGluLeuCysThrLeuArgGluSerVal                              740745750                                                                     AlaLysArgLysAlaThrMetProAspSerSerLeuHisAlaThrSer                              755760765                                                                     SerPheGlnGlyArgArgSerAlaProGluThrIleLeuAlaThrAla                              770775780                                                                     LeuSerMetValArgGluGlnGlnTyrArgGlnGlyLeuGluValMet                              785790795800                                                                  LeuMetAlaGlnGlnProSerLeuLeuLeuArgPheLeuSerIleLeu                              805810815                                                                     ThrArgGluAsnGluAsnAlaTyrSerGluLeuIleGluAsnValGlu                              820825830                                                                     ThrProAsnAspValTrpCysSerValLeuLeuGlnLeuIleGluAla                              835840845                                                                     AlaAlaThrGluAlaGluLysGluValValValGlyValAlaIleAsp                              850855860                                                                     IleLeuSerGluArgAspGlnIleAlaGlnAsnGlyAlaLeuGlySer                              865870875880                                                                  LysLeuThrThrAlaMetArgAlaPheGluArgGlnAlaArgSerGlu                              885890895                                                                     ThrThrSerArgSerPheLeuGlnCysLeuLysAsnLeuIleLysLeu                              900905910                                                                     LeuGlnSer                                                                     915                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "phage DNA"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGTGGCGACGACTCCTGGAGCCCG24                                                    (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "phage DNA"                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TTGACACCAGACCAACTGGTAATG24                                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TCGGGCACTGACGCGGCG18                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "phage lambda gt10 DNA"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CTTATGAGTATTTCTTCCAGGGTA24                                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AACGCTATTATTAGAACAGTT21                                                       (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TGCAGCAGCGGCAGAAGT18                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CAGCCGACGGTAGCTGCGTCCT22                                                      (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ACATAATGGCCTCGTTCACAC21                                                       (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GACTCGCTGCAGATCGATTTTTTTTTTTTTTTTT34                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CGAAGAGACCATGAACAACTT21                                                       (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "DNA"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GACTCGCTGCAGATCGAT18                                                          __________________________________________________________________________

We claim:
 1. A synthetic or isolated protein or protein fragmentselected from the group consisting of:a cytoplasmic protein with anapparent molecular mass of about 100 kDa having the amino acid sequenceof SEQ ID NO:2; and a polypeptide or peptide comprising a fragment ofsaid cytoplasmic protein, wherein said protein or protein fragment isrecognized by anti-Trypanosoma cruzi antisera.
 2. The protein fragmentaccording to claim 1, wherein said protein fragment comprises a firstamino acid sequence starting at amino acid 323 and ending at amino acid520 of SEQ ID NO:2, or a degenerate thereof.
 3. The protein or proteinfragment according to claim 1, wherein said protein or protein fragmentexhibits reactivity with sera from individuals or animals infected withTrypanosoma cruzi.
 4. A composition comprising at least two differentproteins or protein fragments according to claim 1, wherein saidcomposition exhibits reactivity with sera from individuals or animalsinfected with Trypanosoma cruzi.
 5. A reagent for detecting ormonitoring a Trypanosoma cruzi infection, said agent comprising aprotein or protein fragment according to claim
 1. 6. A pharmaceuticalcomposition for the prevention or treatment of infections due toTrypanosoma cruzi, said composition comprising a therapeutically activequantity of a protein or protein fragment according to claim
 1. 7. Asynthetic or isolated molecule indicative of Trypanosoma cruzi, whereinsaid molecule specifically binds (a) anti-Trypanosoma cruzi antisera,and (b) antibodies that specifically bind the protein or proteinfragment of claim
 1. 8. The protein or protein fragment according toclaim 1, wherein said protein or protein fragment specifically bindsanti-PTc100 antibodies.
 9. A synthetic or isolated peptide whichspecifically binds anti-PTc100 antibodies.
 10. The peptide according toclaim 9 wherein said peptide has an apparent molecular mass of about 100KDa.
 11. A synthetic or isolated molecule indicative of Trypanosomacruzi, wherein said molecule specifically binds antibodies that reactwith a cytoplasmic protein with an apparent molecular mass of about 100kDa having the amino acid sequence of SEQ ID NO:2, or a polypeptide orpeptide comprising an immunogenic fragment of said cytoplasmic protein.12. A synthetic or isolated peptide that is encoded by a nucleotidesequence that is identical or degenerate to SEQ ID NO:1, or a fragmentthereof, wherein said fragment is selected from the group consisting ofa first sequence from nucleotide 1232-2207 of SEQ ID NO:1, a secondsequence from nucleotide 1232-1825 of SEQ ID NO:1, and a third sequencefrom nucleotide 1266-2207 of SEQ ID NO:1, wherein said peptidespecifically binds anti-Trypanosoma cruzi antisera.